Integrated bioinformatics analysis for identifying key genes and pathways in female and male patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a common cause of heart failure, and males are more likely to suffer from DCM than females. This research aimed at exploring possible DCM-associated genes and their latent regulatory effects in female and male patients. WGCNA analysis found that in the yellow module, 341 and 367 key DEGs were identified in females and males, respectively. A total of 22 hub genes in females and 17 hub genes in males were identified from the PPI networks of the key DEGs based on Metascape database. And twelve and eight potential TFs of the key DEGs were also identified in females and males, respectively. Eight miRNAs of 15 key DEGs were screened in both females and males, which may be differentially expressed in females and males. Dual-luciferase reporter assay demonstrated that miR-21-5P could directly target the key gene MATN2. Furthermore, Sex differences in KEGG pathways were identified. Both KOBAS and GSEA analysis identified 19 significantly enriched pathways related to immune response in both females and males, and the TGF-β signaling pathway was exclusively identified in males. Network pharmacology analysis revealed that seven key DEGs were potential targets for the treatment of DCM, of which the OLR1 gene was only identified in males, the expression levels of the seven genes were verified by RT-PCR. The above results could offer a novel understanding of sex differences in key genes and pathways in DCM progression.

Network pharmacology analysis. The ingredients and targets of Radix Astragali were retrieved from the TCMSP database (https:// old. tcmsp-e. com/ index. php), and OB ≥ 30% and DL ≥ 0.18 were set as the criteria for screening ingredients. Subsequently, the gene symbols of targets were queried in the UniProt database (https:// www. unipr ot. org/). The intersection of the screened target genes and identified key DEGs were potential targets for treating DCM.
Animal experiments. The cTnT R141W transgenic male mice were purchased from the Chinese Academy of Medical Science. The mice are maintained on a C57BL/6J genetic background and exhibit phenotypic characteristic of human DCM at 4 months of age 22 . Echocardiography showed that the left ventricular end-systole diameter (LVESD) of all the transgenic mice was greater than 2.64 mm. Mice at the ages of 4.5 months were sacrificed and the hearts were excised for RNA isolation. The animal experiment protocol of this study has been reviewed and approved by laboratory animal welfare and ethics committee of Henan University of Chinese Medicine (approval no. IACUC-202305020).
Quantitative RT-PCR. Total  www.nature.com/scientificreports/ ESR1 were detected by the RT-PCR. GAPDH was used as an internal control. The primers are shown in a supplementary table (Table S1).
Statistical analysis. Data were presented as mean ± SD. Statistical analyses were performed by one-way ANOVA and Tukey's test. P-values < 0.05 were considered to be statistically different. P-values were adjusted for multiple testing to control the false discovery rate according to the method of Benjamini-Hochberg, FDR (q-value) < 0.05 was defined as significance cut-off.

Results
Dataset quality assessment. After the expression data of DCM and control samples in dataset GSE141910 was normalized, the distribution of gene expression was observed to be consistent among 332 samples (Fig. 1A).

Identification of DEGs.
Compared to the control group, 794 up-regulated and 322 down-regulated DEGs were identified in the female group, and 753 up-regulated and 363 down-regulated DEGs were identified in the male group.
Identification of key DEGs in the yellow module. WGCNA package of R was adopted for putting genes with similar expression patterns into modules, the power of β = 14 (scale-free R 2 = 0.85) (Fig. 3A, B) was chosen to ensure a scale-free network. Altogether, ten co-expression modules ( Fig. 3C) were identified based on the cluster dendrogram. The correlation between the four groups and ten modules was determined (Fig. 4A, B). Apart from the gray module (that contained unclustered genes), the yellow module correlated significantly with

MiRNAs of the key DEGs.
Based on the GSE112556 microarray data analysis, 13 DEMs were screened and identified in DCM patients. TargetScan, miRbase and DIANA-microT-CDS databases were used to predict target genes of miRNA separately, a total of 1217 target genes of the 13 DEMs were obtained from the three overlapped prediction results, and 15 of the 1217 genes were key DEGs in both females and males in the yellow module. As shown in Table 1, the 15 genes were the target genes of eight miRNAs, of which miR-9-3p targeted five genes (ITIH5, HAPLN1, NRK, CXCL14 and RYR3). In addition, miR-16-2-3p and miR-770-5p collectively targeted BCL11B, whereas miR-21-5p and miR-770-5p collectively targeted MATN2. To verify whether MATN2 is a direct target of miR-21-5p, we fused the MATN2 3'-UTR to a dual-luciferase reporter vector. The luciferase reporter assay results showed that compared to other groups, the relative luciferase activity was significantly repressed in the MATN2 3'-UTR-WT + miR-21-5p group (Fig. 7).

KEGG pathways enriched by KOBAS and GSEA. KEGG pathways analysis for the key DEGs in
the yellow module were conducted by the KOBAS web-based search tool. There were 39 and 37 significantly enriched pathways in the female and male groups, respectively (Fig. 8A, B). Out of these, 35 enriched pathways were common in the female and male groups. Four pathways including PI3K-Akt signaling pathway, NF-κB signaling pathway, antigen processing and presentation, rheumatoid arthritis were unique in the female group, two pathways including transforming growth factor (TGF)-β signaling pathway and calcium signaling pathway were observed to be unique in the male group. GSEA web-based search tool was also used to search enriched KEGG pathways by analyzing the gene expression profile data of the dataset (Fig. 9A, B). GSEA analysis indicated that the genes were significantly enriched in 40 pathways in the female group and 29 pathways in the   (Tables S4 and S5), of which 13 and two pathways were specifically enriched in the female and male groups, respectively. Sex differences in KEGG pathways were analyzed by comparing the results of GSEA and KOBAS using a Venn diagram (Fig. 10). There were 19 common enriched pathways in the female and male groups, including Th17 cell differentiation, Th1 and Th2 cell differentiation, viral protein interaction with cytokine and cytokine receptor, T cell receptor signaling pathway, natural killer cell mediated cytotoxicity, autoimmune thyroid disease, viral myocarditis, primary immunodeficiency, allograft rejection, graft-versus-host disease, intestinal immune network for IgA production, Epstein-Barr virus infection, hematopoietic cell lineage, cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), asthma, protein digestion and absorption, neuroactive ligand-receptor interaction, type I diabetes mellitus. These enrichment terms are significantly associated with immune response. Interestingly, the TGF-β signaling pathway was exclusively identified in the male group, and it was not only enriched by GSEA but also by KOBAS. Five genes (GDF6, FMOD, BMP4, GREM1 and LEFTY2) in the TGF-β signaling pathway were found to have higher levels of expression among males compared with females (Fig. S1).  www.nature.com/scientificreports/ Analysis of Radix Astragali target genes. The Network pharmacology method was adopted to screen target genes of Radix Astragali for treating DCM. Results showed that a total of seven target genes of Radix Astragali (Huangqi) were the key DEGs (Table S6). Six of these DEGs (ESR1, DUOX2, COL1A1, CXCL11, CXCL10 and CD40LG) were identified in both females and males, while OLR1 was only identified in males. Among these genes, CXCL11 and CXCL10 were the hub genes in the PPI networks. Except for DUOX2 and CXCL11, Other genes were targets of the identified TFs. KOBAS analysis indicated that the seven genes were significantly enriched in 40 pathways (Table S7), and these pathways are mainly related to immune response. The seven target genes were up-regulated in DCM, RT-PCR analysis verified that the expression levels of the seven genes were higher in the cTnT R141W transgenic male mouse of DCM model (Fig. 11).

Discussion
Cardiac fibrosis is a common pathological stage of a variety of cardiovascular diseases, and it is a mainly pathological change of DCM. Under pathological conditions, fibroblasts are transformed into myofibroblasts with a stronger ability to secrete extracellular matrix, the formation of myofibroblasts is a key step in the process of cardiac fibrosis 23 . Many factors are involved in regulating cardiac fibrosis, such as TGF-β, matrix metalloproteinases (MMPs), renin-angiotensin-aldosterone system (RAAS), inflammation, and non-coding RNAs (ncRNAs) 24 . The prevalence of cardiac fibrosis is higher in males than in females 25 . Studies have revealed that male mice with myocarditis have a much greater tendency to develop fibrosis and DCM than female mice 26 , more fibrosis has been also detected in male patients with myocarditis compared with female patients 27 . Therefore, Studying the genes involved in the regulation of cardiac fibrosis may be helpful to clarify the sex-based differences in the incidence or pathogenetic mechanisms of DCM. In this study, the PPI networks and TFs of the key DEGs in females and males were analyzed by using Metascape database. These analyses could help to find some key factors involved in the regulation of DCM. We identified 17 hub genes from the PPI networks in both female and male groups. Several genes have been reported to be related to DCM or cardiac fibrosis. For example, HLA-DQ1 polymorphism is involved in the regulation of the immune response and may serve as genetic markers of idiopathic DCM 28 . The expression of CD3D and AGTR2 was increased in the heart following DCM 29,30 , and ventricular-specific expression of AGTR2 promotes the development of DCM and heart failure 31 . CXCL9 and CXCL10 are the main regulators of myocardial inflammatory cell migration and associated to the risk of DCM 32 . Apart from these, LCK, ZAP-70 and APLNR are also found to be associated with DCM 33,34 . TFs are proteins that play important roles in human diseases by binding to specific DNA sequence and regulating the transcription of related genes 35 . Seven common TFs were identified in female and male groups, previous studies indicate that four of these protein genes play important roles in DCM or cardiac fibrosis. Studies show that NFKB1 is involved in the process of cardiac remodeling, and the promoter polymorphism of the NFKB1 gene is associated with the risk of DCM 36,37 . It has been found that deletion of www.nature.com/scientificreports/ endothelial ETS1 in mice following angiotensin II infusion could alleviate cardiac fibrosis 38 . In addition, SP1 or MYB can mediate several miRNAs playing roles in regulating cardiac fibrosis [39][40][41][42][43] .
MiRNAs are a type of ncRNAs with a length of about 22 nucleotides, which negatively regulate gene expression through inhibiting mRNA translation or facilitating mRNA degradation, these molecules have been linked to multiple cardiovascular diseases [44][45][46] . A lot of evidence has shown that miRNAs are involved in regulating the process of cardiac fibrosis by regulating signal pathways or its target genes, thereby affecting the structure and function of the heart 47,48 . For example, studies have shown that miR-1, miR-133a, miR-208a/b and miR-499 participate in regulating cardiogenesis, heart function and pathology 49 . In our study, 13 DCM-related DEMs were obtained by analyzing the miRNA microarray data, and target genes of DEMs were predicted by using three databases. Previous reports have shown sex differences in cardiac miRNA expression 25 , thus miR-21, miR-144, miR-9-3p, miR-770-5p, miR-382-5p and miR-16-2-3p identified in this study may be expressed differentially in females and males. Fifteen target genes were identified in the key DEGs of both female and male groups based on WGCNA. Several studies have shown that some DEMs of the 15 target genes are involved in regulating cardiac fibrosis. For instance, miR-9 inhibits the proliferation of myocardial fibroblasts and the expression of collagen by inhibiting the expression of the target gene PDGFR-β or TGFBR2, and exerts an inhibitory effect on the fibrosis of myocardial fibroblasts 50,51 . MiR-144 has been found to have potent effects on reducing infarct size www.nature.com/scientificreports/ and border zone fibrosis in myocardial infarction model 52 . MiR-21 expression level is significantly increased in the failing heart, silencing of miR-21 can alleviate interstitial fibrosis and cardiac dysfunction by treating mice with a specific antagomir 53 . MiR-21 has also been revealed to be up-regulated in human left ventricular tissues of DCM 54 , which is in accordance with our analysis. The identified up-regulated target gene MATN2 was proved to be a direct target of miR-21-5p, it has been reported to be highly expressed in idiopathic DCM or heart failure 55,56 . More importantly, MATN2 could stimulate cardiomyocyte proliferation 57 . In addition, three target genes (ITIH5, NRK, PDE5A) in DCM and RGS4 in heart failure have been reported to be up-regulated, the expression trend of these genes is consistent with our analysis results [58][59][60][61] . These findings suggest that miRNAs and their corresponding target genes can serve as candidate diagnostic biomarkers or drug targets for the clinical treatment of cardiac fibrosis or DCM. KOBAS and GSEA software were used to predict the significantly enriched KEGG pathways. Nineteen pathways, most of which are related to immune response, were identified in both females and males. It has been confirmed that the pathogenesis of DCM is related to autoimmune response 62 . Myocardial damage can be caused by abnormal immune responses, as it triggers inflammation and recruits immune cells to repair the myocardium, the cytokines released by immune cells such as Th17 and Th2 promote remodeling, collagen deposition and fibrosis, leading to heart enlargement, heart failure and arrhythmia 63,64 . The TGF-β signaling pathway was specifically identified in males. The expression of TGF-β has been observed to be increased in DCM patients 65 , studies have found that the cytokine TGF-β plays a vital role in the development of cardiac fibrosis and the regulation of immune response [66][67][68] . MiR-21 can activate TGF-β/Smad2/3 signaling pathway, cardiac fibrosis could be effectively inhibited by blocking this pathway 69,70 . Due to higher levels of gene expression in the TGF-β signaling pathway in males, thus, it is speculated that activation of TGF-β signaling pathway in males may promote cardiac fibrosis and DCM progression. Furthermore, estrogens have been found to possess  www.nature.com/scientificreports/ antifibrotic effects, another reason that males are more likely to develop DCM than females may be due to the lack of estrogen protection 24,25,71 . Radix Astragali is an important drug widely used in traditional Chinese medicine (TCM) prescriptions for the treatment of DCM, and its treatment effect is remarkable 72,73 . The seven key target genes of Radix Astragali were potential targets for treating DCM, especially the hub genes CXCL11 and CXCL10. The pathways significantly enriched by the seven genes further revealed that immune response may be involved in the progression of DCM. Some experimental results have demonstrated the mRNA or protein expression levels of the seven genes. Among the identified seven up-regulated genes, researches have shown that the levels of DUOX2, CXCL10 and CD40LG expression were significantly higher in patients with DCM 32,74,75 , and ESR1 and COL1A1 were substantially upregulated in the mouse model of DCM 22,76 . In addition, the expression levels of CXCL11 and OLR1 have been proved to be elevated in heart failure patients 77,78 . We also verified that the expression levels of the seven genes were up-regulated in a mouse model of DCM by RT-PCR. These findings suggest that the seven genes are likely to play critical roles in the progression of DCM.
The expression trend of many key DEGs identified in this study is consistent with previously reported results, indicating that the database and our data analysis results are reliable.

Conclusion
We analyzed gene expression profile data of female and male patients with DCM to explore sex differences in the pathogenesis of DCM and to reveal potential diagnostic biomarkers for this disease. And we identified the differences in key genes and pathways between female and male patients by using different analytical methods. This study can provide a reference for exploring diagnostic biomarkers of female and male DCM patients and their latent effects on disease development.

Data availability
All datasets are publicly available derived from NCBI databases. All data generated from the analysis process of this study are available from the corresponding author on reasonable request.